Project Summary Sanfilippo disease (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal storage disorder of childhood whose pathologic features are neurologic: slowing of development, severe behavioral problems, progressive cognitive decline, dementia, and decline in motor skills leading to immobility, unresponsiveness, and death. We have focused on MPS IIID caused by a deficiency of alpha-N- acetylglucosamine-6-sulfatase (GNS). Because MPS IIID is rare (1 in a million) and affects the brain (which is difficult to treat) no cure or treatment is available and there are at least three patients in the USA to our knowledge. Dr. Patricia Dickson and Dr. Tsui-Fen Chou (LABioMed) have developed an enzyme replacement treatment (ERT) for MPS IIID. Our strategy proposes to deliver recombinant human alpha-N-acetylglucosamine- 6-sulfatase (rhGNS) intrathecally or intraventricularly to effectively treat the underlying causes of the neurologic symptoms that dominate MPS III pathology. ERTs are not a cure but can have a dramatic effect on the quality of life and patient development. There are several examples of successfully commercialized ERT's (e.g. laronidase (MPS I), idursulfase (MPS II), etc) and Biomarin recently received approval for an ERT for a form of Batten disease, CLN2. Other ERTs for MPS I, II, and IIIB are in Phase I trials. Both the FDA and investors are familiar with ERT and its commercialization path, which will greatly increase the chances of reaching a clinical trial. We have produced rhGNS ~200 g per 750 mL media in CHO cells, purified it to a specific activity of ~100,000 activity units/mg, demonstrated maximal enzymatic activity at pH 5.6, which is closer to lysosomal pH (with low activity at neutral pH), demonstrated good enzymatic activity at 37C and showed it is stable for over one month at 4 ?C in artificial cerebrospinal fluid storage buffer. We demonstrated intracellular uptake of rhGNS in MPS IIID fibroblasts when rhGNS is added to the media and confirmed that radiolabelled HS diminished 33- 65% in MPS IIID fibroblasts treated with rhGNS (to wild-type levels). We have since scaled up our production of rhGNS so that we can study enzyme activity, lysosomal storage reduction, neuropathology and half-life estimation in the recently characterized MPS IIID knock out mouse. After dosing knockout mice with a single dose of ~5.3 ug rhGNS we have observed return of GNS activity to 50% wild-type levels in mouse brain and a statistically significant reduction of markers of lysosomal storage in this model (NAGLU and hexosaminidase acitivity) after 1 day of treatment. Our currently ongoing studies include histology and half- life estimates after repeat dosing in knock out MPS IIID mice. LABioMed has filed a US patent on rhGNS (pending) and Phoenix Nest, Inc. has licensed it. The pre-IND studies we now propose through this program would include transferring production to a GMP facility, optimizing process for scale-up, and characterizing product (aggregation, glycosylation, purity, activity, contamination, etc). We would also perform 2-animal model pharmacotoxicity studies. Additionally, we would also need to perform other activities to satisfy regulatory agency (FDA) requirements such as assay optimization and regulatory strategy. In order to treat 5 U.S. patients with a possible dose of 90 mg every month (based on Sanfilippo A intrathecal sulfamidase doses), we will need to produce 5,400 mg of rhGNS per year to meet U.S. clinical needs.